(1) Field of the Invention
This invention relates to a novel multistep process for purifying carminomycin 1, a known anthracycline antibiotic.
(2) Description of the Prior Art
Carminomycin 1 is a known antitumor antibiotic which has been isolated from the fermentation broth of Actinomadura carminata as described in U.K. Pat. No. 1,426,637 and in J. Antibiotics, 27, 254 (1974). According to the above references, carminomycin is recovered from the culture medium by extraction with an organic solvent such as chloroform or carbon tetrachloride followed by chromatography over silicic acid.
Isolation of carminomycin 1 from an antibiotic complex (figaroic acid complex) produced by fermentation of Streptosporangium sp. strain C-31,751 (ATCC 31129) has been described by our colleagues W. T. Bradner, D. E. Nettleton, Jr. and J. A. Bush in U.S. Application Ser. No. 832,034 filed Sept. 9, 1977; the entire disclosure of that application is incorporated herein by reference. In this process, the fermentation complex is extracted into an organic solvent (e.g. CH.sub.3 OH or a mixture of CH.sub.3 OH and CHCl.sub.3), the organic extract is subjected to mild acid hydrolysis to preferentially form carminomycin 1 and the carminomycin 1 is recovered by evaporation and purified by chromatography over Sephadex LH-20.
A procedure for isolating carminomycin 1 from the fermentation broth of Streptosporangium sp. has been disclosed by M. E. Wall, et al. in J. Amer. Chem. Soc., 97(20), 5955 (1975). In the Wall procedure, the fermentation broth is extracted with methyl isobutyl ketone, the extract is concentrated and the crude fermentation solids are precipitated by addition of Skellysolve B (isomeric hexanes). The crude fermentation solids are then chromatographed on silicic acid using a gradient eluant of 4:1 (v/v) chloroform: acetone containing 5% CH.sub.3 OH with increasing gradients of CH.sub.3 OH up to 50%.
In view of the increasing interest in carminomycin 1 for treatment of human cancers, there is a need for a new commercially feasible purification process which would allow this antibiotic to be prepared in a highly purified form suitable for pharmaceutical use.
Accordingly, it is an object of the present invention to provide a process for preparing highly purified carminomycin 1 from crude or impure carminomycin 1. It is a further object to provide such a purification process which would be economically practical in the production of commercial quantities of carminomycin 1. A still further object is to provide a purification process which is applicable to impure carminomycin 1 derived from either Streptosporangium sp. ATCC 31129 or Actinomadura carminata, or carminomycin 1-producing mutants thereof.
The foregoing objectives have been achieved by the provision according to the present invention of a process for providing the antibiotic carminomycin 1 in purified form from crude fermentation solids containing said antibiotic which comprises the consecutive steps of
(1) slurrying crude fermentation solids containing carminomycin 1, preferably fermentation solids coated onto diatomaceous earth, with methylene chloride and filtering said slurry to recover the filter cake;
(2) washing the filter cake with water;
(3) extracting the filter cake from step (2) with methanol and recovering a solid from the concentrated filtered extract by concentration to dryness or precipitation with an antisolvent;
(4) dissolving the solid from step (3) in equal volumes of the upper and lower phases of the solvent system consisting of methylene chloride, ethanol, carbon tetrachloride and water (2:1:2:1 v/v); said dissolution being accomplished by first dissolving the solid in a 1:1 (v/v) mixture of methylene chloride and ethanol and then adding to this solution ethanol, carbon tetrachloride, solvent upper phase of the system CCl.sub.4 :CH.sub.2 Cl.sub.2 :C.sub.2 H.sub.5 OH:H.sub.2 O (2:1:2:1) (v/v) and water in that order in an amount sufficient to provide a final solvent system as defined above;
(5) subjecting said solution of step (4) to a countercurrent distribution procedure, said procedure comprising the consecutive steps of:
(a) providing a countercurrent distribution apparatus having a plurality of packed solvent columns connected in series, said columns being packed with conventional glass or ceramic column packing material;
(b) filling all except the first column with the upper phase of the solvent system CCl.sub.4 :CH.sub.2 Cl.sub.2 :C.sub.2 H.sub.5 OH:H.sub.2 O (2:1:2:1) (v/v);
(c) filling the first column with the upper phase of the solution from step (4);
(d) dispersing the lower phase of the solution from step (4) through the columns while collecting samples of the effluent from the last column until all of the solvent has been employed;
(e) dispersing the lower phase of the solvent system CCl.sub.4 :CH.sub.2 Cl.sub.2 :C.sub.2 H.sub.5 OH:H.sub.2 O (2:1:2:1) (v/v) through the columns while collecting samples of the effluent from the last column until thin layer chromatographic analysis of the various samples and solutions in the columns indicates cessation of any significant separation of components;
(f) analyzing the samples taken and the solutions from the columns by thin layer chromatography to detect the carminomycin 1-rich solvent fractions;
(g) combining the carminomycin 1-rich solvent fractions and recovering the impure carminomycin 1 solids from said combined fractions;
(6) dissolving the solids from step (5) in chloroform:methanol (17:3) (v/v);
(7) adsorbing the solution from step (6) on silica gel which has been slurried with chloroform:methanol (17:3) (v/v) and adjusted to a pH of about 7-9; and
(8) eluting the components of the impure carminomycin 1 mixture with a solvent system consisting of chloroform:methanol (17:3) (v/v) to produce purified carminomycin 1.
The present invention may advantageously be employed with any source of impure carminomycin 1 but is most useful for purification of crude fermentation solids derived from culture broths of carminomycin 1-producing strains of Actinomadura carminata or Streptosporangium sp. (i.e. Streptosporangium sp. ATCC 31129 or a carminomycin 1-producing mutant thereof). Such crude fermentation solids may be obtained from the fermentation medium by known methods, e.g. by extraction of the filtered broth or whole broth with a water-immiscible organic solvent such as methylene chloride or methyl isobutyl ketone and recovery of the carminomycin 1-containing fermentation solids from the organic extract by concentration to dryness or by concentration and precipitation with an appropriate antisolvent, e.g. diethyl ether, benzene or a liquid aliphatic hydrocarbon such as n-hexane, n-heptane or isomeric hexanes (Skellysolve B) in the case of the antibiotic complex (designated figaroic acid complex) from Streptosporangium sp. and ethyl acetate, benzene, chloroform, diethyl ether or petroleum ether in the case of the antibiotic complex from Actinomadura carminata. The purification procedure of the present invention is most efficient when the crude carminomycin 1 is recovered from the fermentation broth coated onto diatomaceous earth (filter aid) by a procedure such as illustrated in Preparation A-3 below.
The crude carminomycin 1-coating fermentation solids are commonly contaminated with filter aid (diatomaceous earth), fats, sugars, defoaming agents and other biologically inactive materials associated with the fermentation and preferred recovery steps. To separate such contaminants from the biologically active components, the fermentation solids are subjected to a solvent extraction and wash procedure as described in steps 1-3 above. Thus, the crude carminomycin 1 solids are first slurried with methylene chloride. The slurry is then filtered and the filter cake washed with methylene chloride. Advantageously this procedure is repeated one or more times. The filter cake from the methylene chloride wash is next washed with water one or more times to remove water-soluble impurities such as sugars. The washed filter cake is then extracted with methanol, the methanol extract filtered and concentrated and the carminomycin 1-containing solids recovered from the concentrated extract by precipitation with an appropriate antisolvent (e.g. diethyl ether, benzene or a liquid aliphatic hydrocarbon or mixture of hydrocarbons such as n-hexane, n-heptane or isomeric hexanes) or by evaporation to dryness.
Following steps (1) through (3), the impure carminomycin 1 solids recovered from the methanol extract are subjected to a countercurrent distribution procedure to separate the antibiotic mixture into component groups closely related into polarity. Separation by countercurrent distribution may be carried out with any conventional apparatus, e.g. a Craig apparatus. In the production of larger than laboratory quantities of purified carminomycin 1, however, it is preferred to employ a series of packed solvent columns such as are commonly used in large scale pilot plant or industrial extractions.
An apparatus found to be especially advantageous for the purposes of the present invention may be constructed by connecting in series a plurality (preferably at least 5) of packed solvent columns, e.g. Glenco solvent columns distributed by Glenco Scientific Inc., Houston, Texas. The columns are connected, e.g. with Teflon or polyethylene tubing, from the bottom of one column to the top of the next. While good results have been obtained with five solvent columns, acceptable separation can be achieved with any number of columns from two to several hundred. The columns are filled with conventional glass or ceramic packing material such as Raschig rings or Berl saddles.
The impure carminomycin 1 from step (3) of the process is dissolved in a two phase (equal volumes of each phase) solvent system consisting of methylene chloride, ethanol, carbon tetrachloride and water in the volume ratio of 2:1:2:1, respectively. The method of dissolution, however, is critical for obtaining significant amounts of the solid in solution in a practical time period. Thus, the solid is first dissolved in a 1:1 v/v mixture of methylene chloride and ethanol and there is then added to this solution ethanol, carbon tetrachloride, solvent upper phase of the system CCl.sub.4 :CH.sub.2 Cl.sub.2 :C.sub.2 H.sub.5 OH:H.sub.2 O (2:1:2:1 v/v) and water in that order in amounts sufficient to provide a final solvent system of equal phases as defined above. The phases from the solution are separated and filtered for use in the countercurrent distribution procedure.
In carrying out the countercurrent separation using the preferred apparatus described above, all of the solvent columns except the first are filled with upper phase of the solvent system CCl.sub.4 :CH.sub.2 Cl.sub.2 :C.sub.2 H.sub.5 OH:H.sub.2 O (2:1:2:1 v/v). The first solvent column is then filled with the upper phase of the sample solution as prepared above. Next, lower phase of the sample solution is pumped into the top of the first column on through the other columns top to bottom and out the bottom of the last column. The pumping rate is not critical, but a convenient rate has been found to be about 100 ml. per 24 minutes. Effluent from the last column is collected with a fraction collector for thin layer chromatographic analysis. After all the sample solution lower phase has been used, lower phase of the solvent system CCl.sub.4 :CH.sub.2 Cl.sub.2 :C.sub.2 H.sub.5 OH:H.sub.2 O (2:1:2:1 v/v) is pumped over the columns until monitoring of the effluent fractions and column solutions by thin layer chromatography indicates that separation of the antibiotic components has been effectively completed.
A suitable thin layer chromatographic system for analyzing the various effluent fractions and the solutions in the solvent columns consists of silica gel plates developed with a chloroform:methanol:formic acid (85:15:1 v/v) solvent system. R.sub.f values of the antibiotic components may be determined on such plates by viewing visually and under 2537 A ultraviolet light. Fractions are combined based on the color bands seen with the above assay and the combined fractions are then concentrated in vacuo at temperatures not exceeding 30.degree.-35.degree. C. The solids from the carminomycin 1-rich fractions are then used in the final chromatographic steps (6) through (8).
After dissolving the partially purified carminomycin 1 obtained in the countercurrent separation procedure in chloroform:methanol (17:3 v/v), this solution is subjected to column chromatography over silica gel using 17:3 (v/v) chloroform:methanol as the eluting solvent. The silica gel used in the column is acid washed to remove iron, dried (.ltoreq.2% water content), slurried in eluting solvent and adjusted to a pH of between about 7-9 by basification of the slurry, e.g. with concentrated ammonium hydroxide. Eluant fractions from the column are collected and analyzed by the above-described TLC system to determine the fraction(s) which are purified carminomycin 1. The solvent fraction(s) containing the desired purified product may then be treated by conventional means such as concentration or lyophilization to give carminomycin 1 which is sufficiently pure for pharmaceutical use.
The following examples serve to illustrate the invention without limiting it. "Skellysolve B" is the trade name for a petroleum ether fraction of b.p. 60.degree.-68.degree. C. consisting essentially of n-hexane and sold by Skelly Oil Co. All temperatures referred to herein are in degrees Celsius. The abbreviation "MIBK" refers to methyl isobutyl ketone.